ES2306916T3 - PROCEDURE FOR THE SYNTHESIS AND MODIFICATION OF BIOCATALITIC SELECTIVE PEPTIDES, MIMETIC SUBSTANCES OF PEPTIDES AND PROTEINS. - Google Patents

Abstract

Method for synthesis and/or selective N-terminal modification of peptides, peptide mimetics and/or proteins (A). Method for synthesis and/or selective N-terminal modification of peptides, peptide mimetics and/or proteins (A) comprises: (1) preparing an amino component (I) containing at least one amino acid (aa); (2) preparing a carboxy component (II) that has a leaving group on carboxy and contains at least one aa, or a marker or reporter group; and (3) reacting (I) and (II) in a medium that contains at least one ionic fluid, in presence of protease, peptidase and/or hydrolase, so that (I) and (II) become linked through a peptide bond, with cleavage of the leaving group.

Description

Procedure for synthesis and modification selective biocatalytic of peptides, mimetic substances of peptides and proteins

The present invention relates to a procedure for enzymatic synthesis and / or selective modification of peptides, mimetic substances of peptides and / or proteins through the use of ionic liquids as well as the use of ionic liquids as the exclusive means for the reaction, or in combination with water and / or organic solvents to prevent formation of proteolytic side reactions and hydrolytic

The synthesis and selective modification of peptides, mimetic substances of peptides and proteins, is of increasing importance in the systematic study of the structure-function relationships of polypeptides as functional genetic products and leads decisively to the discovery of new effective therapeutic substances (see H. -D Jakubke, Peptide: Chemie und Biologie (" Peptides: Chemistry and Biology "), Spektrum Akademischer Verlag, Heidelberg, Berlin, Oxford, 1996). Essential problems of its synthesis or its selective modification are, however, the lack of selectivity and universality of chemical processes and the limitation of substrates or, respectively, the occurrence of numerous secondary reactions in the use of enzymes as catalysts.

Fundamentally, they can be used for peptide synthesis, peptide mimetic substances, and proteins, the chemical methods developed in the chemistry of peptides These are, however, subject to considerable limitations when the complexity of the product increases. While that peptides with an average chain length of 50-60 amino acids are directly accessible by solid phase peptide synthesis, another extension of the chain, because the coupling performance is never quantitative, often leads to the accumulation of a large number of secondary products, which lead both to a decrease in synthesis performance as well as to hinder or respectively prevent the purification of the desired product. The current methods for polypeptide synthesis or respectively of longer proteins, are based on fragment condensation of synthetically prepared peptides but, nevertheless, the binding of completely protected peptide fragments, it is only possible in exceptional cases, because of the solubility often Very small, of the educts.

The methods developed based on the molecular clamp eraser first proposed in 1953 for CN-chemical linkages of unprotected peptide fragments (T. Wieland et al ., Annalen (" Annals ") 1953 , 583, 129; M. Brenner et al ., Helv. Chim. Acta 1957 , 40, 1497), from the capture of amino- and thio- (DS Kemp et al ., J. Org. Chem . 1975 , 40, 3465; N. Fotouhi et al. ., J. Org. Chem . 1989 , 54, 2803) of natural chemical ligation (M. Schnölzer, SBH Kent, Science (" Science ") 1992 , 256, 221; PE. Dawson et al ., Science (" Science ") 1994 , 266,776) or also the method of aldehyde (C.- F. Liu, JP Tam, Proc. Natl Acad. Sci USA 1994 , 91, 6584), actually take place selectively, but nevertheless, they need to be carried out , very specific N- or C-terminal amino acid radicals, so that their usability depends on the sequence specificity requirement. In the case of the currently preferred native chemical ligation, the union of a synthetic peptide with a thioester group with a C-terminal takes place with a second peptide or protein which must contain a cysteine radical with an N-terminal. By taking advantage of the knowledge of Protein mutation The native chemical ligation evolved to a binding with the intein-mediated protein (" expressed protein ligation ")], EPL; (See among others TW- Muir et al ., Proc. Natl. Acad. Sci. USA 1998 , 95, 6705; GJ Cotton et al ., J. Am. Chem. Soc . 1999 , 121, 1100), in which the thioester group of the carboxylic component of a recombinant protein, which is fused with an intein competent for cleavage, and is formed by a thiolitic cleavage. Together with the need for a cysteine radical in the N-terminal of the amino component, there is another general disadvantage in the non-exclusive partial epimerization of the amino acid radical of the C-terminal, since the thioester formed (at least when thiophene is used as a catalyst ) can not only be attacked after trans-esterification but also nucleophilically directly by the α-amino terminal groups of the added amine component.

Synthetic catalytic processes have the advantage of greater flexibility with respect to the synthetic formation of peptides with preparative importance, although in Nature there is no known, at least until now, any universal peptide ligase with preparative importance. Thus, the catalytic antibodies show CN-ligase activity (see among others, PG Schultz, RA Lerner, Science (" Science ") 1995 , 269, 1835; G. MacBeath, D. Hilvert, Chem. Biol . 1996 , 3, 433 , DB Smithrub et al ., J. Am, Chem. Soc . 1997 , 119, 278), also as synthetic peptide ligases based on a coiled-coil ("rolled") motif of GCN4 (K. Severin et al ., Nature (" Nature ") 1997 , 389, 706) or respectively on a peptide matrix composed of a strongly acid coiled-coil ("rolled") peptide (S. Yao, J. Chmielewski, Biopolymers (" Biopolymers ") 1999 , 51 , 370). In all these cases, it is undoubtedly an interesting starting point for the design of peptide ligases, which in any case require binding, special requirements, and whose general usability is therefore very limited. It is known that proteases can be used as catalysts for enzymatic peptide synthesis, for example papain proteases and chymotrypsin for the synthesis of ovoid peptides (W. Kullmann, Journal of Biological Chemistry ( "Journal of Biological Chemistry") 1980 vol 255, No. 17, 8234-8238). The use of reverse potential catalysis of peptidases (see among others W. Kullmann Enzymatic Peptide Synthesis ( "Enzymatic Peptide Synthesis"), CRC Press, Boca Raton, 1987; H.-D. Jakubke, Enzymatic Peptide Synthesis ( " Enzymatic synthesis of peptides ") in: The Peptides: Analysis, Synthesis, Biology , (" Peptides: Analysis, Synthesis, Biology ") vol. 9 (editors: S. Udenfriend, J. Meienhofer), Academic Press, New York, 1987 , Chapter 3) really offers the important possibility of enzymatically binding peptide segments under special conditions, although it does not guarantee the irreversibility of the formation of special bound peptides, nor does it exclude a priori unwanted proteolytic cleavages in the bound segments or respectively in the product Finally, when there are potential cleavage sites for the peptidase used. Although by reengineering different peptidases, such as p. For example, subtilisin, the potential for catalysis for the formation of peptide bonds can be improved and can also be demonstrated by demanding fragment condensations (see, among others, DY Jackson et al ., Science (" Science ") 1994 , 266, 243 ), the disadvantages described above cannot be eliminated. The concept of mimetic substances of the substrate developed from the peptide and protein segments for CN- junctions (F. Bordusa et al ., Angew. Chem . 1997 , 109, 2583; Revision: F. Bordusa, Braz. J. Med. Biol. Res . 2000 , 72, 469) really has the advantage of irreversibility, although nevertheless, it also needs the use of synthetic proteolytic variants, proteolytically inactive, to prevent competitive cleavages within the biopolymers to be bound. WO 02/26772 describes a process for the selective catalytic modification of peptides and proteins with the use, among others, of trypsin and chymotrypsin.

For modification of peptides, peptide mimetics and proteins, and still have played an important role in juegan- protein research, chemical procedures (see T. Imoto, H. Yamada, Chemical Modification ( "Chemical modification") ., protein Function a practical approach ( "Function of protein a practical method.") (TE Creighton editors) pages 247-277, IRL Press, 1989; GE means, RE Feeney, chemical Modification of Proteins ( "chemical Modification proteins "), Holden-Day, 1971 ). Thus, despite the rapid progress of the NMR technique, which in the last decade has made possible a complete subordination of the signals and thus a clarification of the three-dimensional structures of the proteins up to 150-200 structural amino acids (without modification), the chemical modification is also a tool for determining the spatial structure in solution, since large proteins are not accessible to structural analysis by NMR, so they need a structural analysis by Roentgen rays of protein crystals , which in many cases cannot be obtained.

Since the preferred objectives of the selective modifications are the N-terminal α-amino groups, the ε-amino groups allow the ubiquity of the lysine radicals present in proteins and peptides, and no selective introduction of marker and informational groups into the terminal N. The chemical acylation reactions are carried out with anhydrides or preferably with active esters, such as p. eg, N-hydroxysuccinimide esters or 4-nitrophenyl esters, whereby other side chain functions can also react with proteinogenic amino acid radicals and thereby exclude a selective Nα modification. Only the specificity of the phenylacetyl radical enzymatically introduced by penicillin acylase in the reversal of the native effect as a protective group for amino acids in the context of peptide synthesis was determined, (R. Didziapetris et al ., FEBS Lett . 1991 , 287, 31) and by the same enzyme it is cleaved again (see review: A. Reidel, H. Waldmann, J. prakt. Chem . 1993 , 335, 109). Apart from this direct introduction of protection groups, only those methods based on the catalysis of peptidases on a transfer of amino acid derivatives or peptides already labeled at the N-terminal, with specific amino acid esters of the peptidase, in the P_ position were described. 1}, and do not necessarily present any irreversibility. An exception to these is the original concept of substrate mimetic substances developed for CN- linkages of peptide and protein segments (F. Bordusa et al ., Angew. Chem . 1997 , 109, 2583; Revision: F. Bordusa, Braz. J. Med, Biol. Res . 2000 , 72, 469). This method really has the advantage of the direct and selective introduction of marker and informational groups, but nevertheless, it needs, as mentioned above, the use of inactive, proteolytic, synthetic protease variants, such as biocatalysts, to prevent them from taking place. Competitive cleavages of biopolymers to be marked.

The suppression of hydrolytic and proteolytic side reactions of hydrolases used for the synthesis and modification of peptides, mimetic substances of peptides and proteins, can be carried out together with selective enzymatic engineering also by manipulations in the reaction medium. The literature describes the use of mixtures of a single phase of water and organic solvents, of analogous biphasic systems in the case where water and organic solvent are not miscible, of pure organic solvents with practically none or only a small part of water, of frozen or low-temperature aqueous or organic systems, of supercritical solutions and heterogeneous mixtures with virtually no or very little solvent (see, among others, W. Kullmann, Enzymatic Peptide Synthesis (" Enzymatic Peptide Synthesis ") , CRC Press, Boca Raton, 1987; H.-D. Jakubke, Enzymatic Peptide Synthesis (" Enzymatic Peptide Synthesis "), in: The Peptides: Analysis, Synthesis, Biology (" Peptides. Analysis, Synthesis, Biology ") , vol 9, (editors: S. Udenfriend, J. Meienhofer), Academic Press, New York, 1987, chapter 3). In any case, practically all of these methods act by dramatically decreasing the enzymatic activity or, respectively, the stability and in part require considerable expenditure on apparatus. To this it should be added that only few have been investigated in general in terms of their usability for the synthesis and modification of long chain biopolymers. But neither in these cases has any of these procedures been able to accredit to be routinely used to determine the required efficiency and universality.

A new class of solvents is represented by salts, which have a low melting point. For these solvent systems, also designated as ionic liquids, a stabilizing action on proteins and enzymes could be found in initial studies (review: CM Gordon, Appl. Catal. A: Gen. 2001 , 222, 101). It was seen that the use of the ionic liquid ethylammonium nitrate favored the refolding of a reduced / denatured chicken albumin protein (lysozyme) and that it may hinder the aggregation of the denatured protein (CA Summers, RA Flowers, Protein Science ("Science of the proteins ") 2000 , volume 9, nº 10, 2001-2008).

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It is also known that ionic liquids have good properties as peptide and protein solvents and can therefore be used for coating matrices in UV-MALDI analysis (DW Armstrong et al ., Analytical Chemistry ("Analytical Chemistry"), United States 2001 , Volume 73, No. 15, 3679-3686).

It is also known that lipases are not inactivated by the action of ionic liquids. Certain ionic liquids show polarities, which are similar to polar organic liquids. It was found that 1-phenylethanol-catalyzed lipase acetylation is as rapid in ionic liquids as with toluene (S. Park, Journal of Organic Chemistry 2001 , volume 66, No. 25, 8395-8401). In simple model reactions with lipases and galactosidases it was also seen that these liquids have a positive effect on the speed of the reactions and partly also on the selectivity of the enzymatic reactions (U. Kragl et al ., Chimica Oggi (" Chemistry today ") 2001 , 19, 22, TL Husum et al ., Biocatal. Biotrans. 2001 , 19, 331; SH Schofer el al ; Chem. Commun., 2001 , 425). In the example of a simple substrate of an amino acid ester it could also be demonstrated that also serine, chymotrypsin and subtilisin proteases in reaction systems with a high proportion of said liquids are enzymatically active and catalyze both the hydrolysis of the ester and its transesterification (JA Laszlo, DL Compton, Biotechnol., Bioeng . 2001 , 75, 181; TL Husum et al . Biocatal. Biotrans, 2001 , 19, 331). In view of the synthesis of Z-Asp-Phe-OMe from Z-Asp-OH and H-Phe-OMe by means of the thermolysin metalloprotease, the important suitability of ionic liquids for protease catalyzed binding could also be demonstrated of two amino acids with the synthesis conditions controlled in equilibrium (M. Erbeldinger et al ., Biotechnol. Prog . 2000 , 16, 1131). Until now, on the contrary, it is completely unknown whether such liquids can be selectively bound to peptide fragments by proteases in a series of genetically controlled reactions, and if under these conditions a selective introduction of informational groups and markers at the N-terminus of The peptides and proteins are catalyzed by proteases. Similarly, the influence of ionic liquids on the extent of proteolytic side reactions in the educts as well as in the hydrolytic side reactions on the ester substrate used is not clear.

The present invention is intended to set a procedure for enzymatic synthesis and modification of peptides, mimetic substances of peptides and proteins, which solve the inconveniences of the procedure described in the state Current technique. The present invention also has purpose of setting up a procedure in which it has place a sequence dependent synthesis, in particular the ligation and modification of terminal N, without taking place proteolytic and hydrolytic side reactions in educts or respectively in the reaction products, regio- and stereo-selectively

The problem is solved according to the invention. by a procedure for the synthesis of peptides, substances mimetics of peptides and / or proteins and / or for modification N-terminal selection of peptides, mimetic substances of peptides and / or proteins,

with the following steps:

a) preparation of an amino component, in where said amino component has at least one amino acid,

b) preparation of a carboxylic component, in where said carboxylic component has a labile group in the group carboxyl, and the carboxylic component has a bond with at less an amino acid or a compound with at least one group bookmark or informational,

c) reaction of the amino component and the carboxylic component in a reaction medium which has one or various ionic liquids, in the presence of a protease, peptidase and / or hydrolase, where between the amino component and the component carboxylic, a peptide bond is formed with group cleavage labile

In a preferred version it is provided that the method according to the invention further comprise the following Steps:

d) isolation or enrichment of the peptide, mimetic substance of peptide and / or protein obtained, by procedures already known.

In addition, the present invention relates to use of ionic liquids as exclusive solvents or in combination with water and / or organic solvents for synthesis and / or N-terminal modification of peptides, mimetic substances of peptides and / or proteins. The present invention also relates to use of a protease, peptidase and / or hydrolase for synthesis and / or N-terminal modification of peptides, mimetic substances of peptides and proteins, wherein the peptide, the mimetic substance of peptide and the protein or its species labeled in the N terminal are obtained by ligation of an amino component and a component carboxylic, and the carboxylic component has a labile group.

Other versions are deduced from the Secondary claims and the following description.

According to the invention, they are comprised between peptides, amino acid condensation products with approximately 2-10 amino acids. Between the Polypeptides are understood according to the invention, the products of amino acid condensation with approximately 10-100 amino acids and the concept of protein is used according to the invention for condensation products of amino acids, which have more than about 100 amino acids, in where the passage between both concepts is fluid in the literature.

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With the name of peptide mimetic substances, those compounds according to the invention are designated which mimic or antagonize the biological activity of a peptide without properly having the classical structure of a peptide formed exclusively of encoded amino acids. Examples of peptide mimetic substances are in conjunction with completely non-peptide organic compounds (eg morphine or naloxone respectively formed from cyclo-aliphatic and aromatic structures) and also those having modified amino acids (eg amino acids N -, α- and β-alkylated, amino acids of α to β carbon atoms and cyclic amino acids N-Cβ; peptides with modified side chains such as for example α-, β-dehydrogenated amino acids, nitrotyrosine, etc.), as well as cyclic substances analogous to peptides (cyclisation of the N terminal with the C terminal or respectively amino acid side chains, cyclisation of the C terminal with amino acid side chains or respectively cyclisation of amino acid side chains with side chains of amino acids) and peptides with modified peptide bonds such as thioamides, ketomethylenes, ethylenes, methylenamines or tam Well retro-inverse-derivatives, among others. Retro-inverse-derivatives are compounds with a backbone peptide structure RC-NH-CO-C-R ', in which the position of the amino function and the carboxyl function is exchanged compared to normal peptide junctions, while normal peptide bonds have the structure
RC-CO-NH-C-R '.

Ionic liquids are, unlike melts of salts, salts that melt at low temperatures (<100 ° C), which consist exclusively of ions (Lit S. e.g., T. Welton Chem. Rev. 1999 , 2071 -2083). According to this definition, water is not an ionic liquid either. Characteristic peculiarities are its low symmetry, few intermolecular exchanges and a good load distribution. Typical cations contain quaternized heteroatoms such as quaternized ammonium ions or quaternized phosphonium ions. Subgroups are p. eg, N-alkylated imidazolium ions such as 1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, N-alkylated pyridinium ions such as 4-methyl-N-butyl-pyridinium or respectively ammonium and phosphonium analog ions replaced. Typical anions can be both inorganic and organic in nature, such as p. eg, chloride, bromide, chloroaluminate, nitrate, benzosulfonate, triflate, tosylate or also tetrafluorborate.

From the phenomenon observed so far in the current state of the art, that the complete replacement although also in part, of water as a reaction medium by solvents inert to the reaction, leads to a loss of activity until enzyme inactivation, the present invention, it was based on the surprising discovery that it is possible to join a carboxylic component provided with a labile group, wherein the carboxylic component represents a peptide, a mimetic substance of peptides, a protein or respectively a marker or informational group, catalytically catalyzing with a high speed of synthesis and selectivity, with the use of liquids ionic as exclusive solvent, or in combination with water and / or organic solvents as a reaction medium, with one component amine which preferably represents a peptide, a substance peptide mimetics, and a protein.

It is also surprising that the reactions side effects typically present, such as hydrolysis of the junction between the carboxylic component and the labile group, as well as the proteolysis of the peptide junctions corresponding to the specificity of the enzyme, they do not take place practically in the educts or reaction products respectively. He carboxylic component is typically provided with the labile group, so that the labile group, of the ester, thioester or amido type is linked to the carboxyl function of component C terminal carboxylic. The process according to the invention is based on the surprising discovery that ionic liquids or mixtures of them unlike virtually all others organic solvents, have a favorable influence on the enzyme synthesis activity, and at the same time hinders virtually unwanted side reactions typically propitiated by the water solvent. Through the combined use of an ionic solvent with the components carboxylics, which contain the specific labile groups of enzymes, independence of activity can also be achieved of synthesis, of the original specificity of the substrate of the enzyme, which decisively increases the breadth of employment for the Synthesis of the procedure

It has been discovered, that all proteases of serine and cysteine investigated show the behavior described previously and therefore are particularly appropriate, within the framework of the method according to the invention, for the synthesis and peptide modification, peptide mimetic substances, and proteins Other appropriate enzymes can be obtained within the framework of selection processes through the use of model reactions appropriate, as can be done with the teaching base technique disclosed.

Within the framework of such a selection process, proceeded so that the synthesis activity of proteases, peptidases and / or hydrolases in ionic liquids or mixtures thereof, by means of model synthesis reactions. For this, a component is incubated in the simplest case carboxylic compound of amino acids (carboxylic components or mimetic substrate substance) with an amino component, in the simplest case an amide of an amino acid, but preferably a peptide and protease, peptidase or hydrolase test. The tolerance of ionic liquids to the enzyme, is shown by product training in the course of the next phase of incubation. The product formation itself can be analyzed by example, by HPLC or other chromatographic methods of separation.

According to the invention, they are preferred as proteases, cysteine proteases or serine proteases. But nevertheless, basically all other known classes of proteases, also aspartate proteases or metalloproteases. Like other hydrolase groups, they are particularly in question, Lipases or esterases. As peptidases (EC 3.4.11-3.4.19) can also be used in a manner general according to the invention, known subgroups of peptides. For the definition of hydrolases, peptidases and proteinases see in particular, Römpp Chemielexikon, 9th edition, 1989-1992.

Another advantage of the procedure according to the invention is based on the "regal" specificity of the enzyme employed and the non-existent risk of racemization, in comparison With most chemical procedures. In this sense it is advantageous than educts with chiral centers and other functions acylates can be used without temporary blocking measures and experimentally expensive selective and conducive to reactions Additional secondary. They are exceptions, only the necessary introduction in some cases of terminal protection groups N in the carboxylic component, which is necessary in particular, when the N-terminal sequence of the carboxylic component has greater specificity for the enzyme than the terminal sequence N of the amino component. In addition, they do not exist on the contrary, for chemical selective methods, virtually no limitations with respect to the sequence of the educts that appear in the reaction.

According to the invention, the proteases, peptidases and / or hydrolases, as enzymes. These present preferably a selectivity or specificity for the labile group and / or certain amino acids or amino acid regions of the carboxylic component The labile group and / or these amino acids or amino acid regions may be preferred known compounds according to the invention of the enzyme used naturally. May be treated according to the invention preferably also of compounds structurally similar (mimetic substances of substrate).

The concepts of amine component and component carboxylic, as used herein, are defined in relation to to the polypeptide to be synthesized. Thus, the concept of component Aminic refers to a chemical compound that has at least one available amino group, which reacts with a carboxyl group or a derivative thereof or respectively a carboxyl group that is derivatized with a labile group, with bond formation peptide In the amino component it is preferably a amino acid, more preferably, of a polypeptide or protein. In the latter case, the polypeptide or protein has both a amino end as a carboxyl end. The carboxyl end is in addition, either unprotected or protected. To avoid a reaction with another reactive group such as the amino group of another molecule of the amino component, the carboxyl group of amino component is typically and preferably present in form not activated. It is further preferred that the function Nα-amino of the amino component is without protect, so this function N? -Amino reacts with the function carboxyl of the carboxylic component.

In the carboxylic component, it is about preference of a marker or information group provided with a carboxyl function, or of an amino acid or, more preferably of a polypeptide or protein. The carboxyl function or the carboxyl group of the carboxylic component which reacts with the amino group, by general rule the amino group of the N-terminal of the amino component with formation of a peptide bond, typically and preferably active

It is preferable, according to the invention, that the group labile of the carboxylic component is selected from the group consisting of -O-alkyl-, -O-aryl-, S-alkyl-, radical -S-aryl-, -NH-alkyl-, -NH-aryl-, -N, N-dialkyl-, -N, N-diaryl- and the radical -N-aryl-N-alkyl, unsubstituted or substituted, where also preferably, the labile group of the carboxylic component is substituted with one or various carboxyl radicals, sulfonic acid radicals or sulphonate The alkyl concept includes in this relationship also cycloalkyl and heterocycloalkyl. How heteroatoms enter particular issue N, O and S. Cycloalkanes are preferred or heterocycloalkanes with 5-6 carbon atoms in the ring. Among the alkyl radicals are included the radicals n-alkyl and branched alkyl radicals according to the invention, preferably n-alkyl radicals with 1-5 atoms of C.

The aryl concept comprises in this relationship, in particular substituted and unsubstituted phenyl, which is of preference substituted with guanidino and / or amidino in the ring of phenyl. The aryl concept here also includes annular systems ringed, preferably biphenyl, and not ring systems ringed like naphthyl, which can be again from preference substituted with guanidino and / or amidino groups, and heteroangous systems such as quinoline or isoquinoline. He aryl concept here also includes heteroaromatic compounds with 5-6 atoms in the ring, where one or several ring carbon atoms, are preferably substituted by N, O, and / or S. Examples, are the radicals pyridin-, thiophene-, furan-, pyrazole- or imidazole-.

It is particularly preferred that the labile group of the carboxylic component be a radical 4-guanidinophenyl, 4-amidinophenyl, 4-guanidinophenylthio or 4-amidinophenylthio or a structurally compound counterpart to them.

In the carboxylic component, the labile group form with the carboxyl group of the carboxylic component of preferably an ester or an amide, preferably in the group C-terminal carboxylic of the carboxylic component. As already mentioned, the enzymatic activity of the enzyme by specific recognition of the labile group of the end end, so that also the peptide fragments or the marker groups and informants come together instead of educts, with specific amino radicals of enzymes by protease,  hydrolase or peptidase.

For arginine specific proteases, such as trypsin, a mediating effect of the specificity for the ester labile group 4-guanidinophenyl. An analogous function is observed for the amidinophenyl ester. In addition, based on homology structural analogues of 4-guanidinophenylthioester- and 4-amidinophenylthio-ester possess a similar effect and also have advantages in chemical synthesis. Structural homologues of these compounds also enter consideration as labile groups mediating the specificity

As structurally homologous compounds derivatives of these compounds serve with a basic group, such as for example, the amino-, amidino-, guanidino- and imino- groups, which interact with amino acid residues of the protease determinants of specificity, that is, in particular, said amino acid radicals, which directly or indirectly with the substrate enter into reciprocal action, respectively those that they influence the catalytic reaction and which have a body aliphatic or aromatic base with for example a chain length between one and six methylene units or benzene basic bodies, naphthalene or indole, between the specific basic group and the function ester or respectively amide as a binding element between the group carboxyl of the carboxylic component and the labile group. In sense figured this also applies to enzymes with a primary specificity for glutamic acid or respectively aspartic acid, such as protease V8, with the difference that instead of the basic groups in the bodies basic aliphatic or aromatic, acidic groups are attached as carboxyl and sulfonic acid groups. Similarly, esters or the amides, which consist only of the aforementioned bodies basic that does not have any basic or acidic group, they constitute carboxylic components for enzymes with a preferred specificity for hydrophobic amino acid radicals, such as chymotrypsin and subtilisin.

According to the invention, it is preferred to adapt the group labile to the specificity of protease, peptidase and / or hydrolase employed

In another preferred version, the component carboxylic containing the labile group has the following structure:

Y- (Xaa) n -R

in where

Y = a protection group of N-terminal, or is an H,

Xaa = an α-amino acid, or is it any β-amino acid, or is it a derived from them, or is a marker or informational group,

R is a labile group, in particular it is a labile group chosen from the group comprising -O-alkyl-, -O-aryl-,
-S-alkyl-, radicals -S-aryl-, preferably 4-guanidinophenyl-, 4-amidinophenyl-, 4-guanidinophenylthio-, radicals
4-amidinophenylthio-, unsubstituted and substituted, which may be substituted in each case by sulfonic acid groups or sulfonates, as well as structural homologs thereof,

n is an integer from 1 to 1000, of 30 to 500 preference, and more preferably, 30 to 250.

The concepts alkyl and aryl have the meaning defined above, and understand as above also, cycloalkanes, heterocycloalkanes, ring systems ringed and ring systems not ringed as well as the versions Preferred cited above.

It is also preferred that the component carboxylic be a marker or informational group, chosen from the fluorescent markers such as fluorescein, rhodamine, tetramethyl rhodamine acid 2-aminobenzoic acid, which contain carboxyl groups; Isotope markers containing carboxyl groups such as amino acids containing C 13 -, N 15 - and O 17 - or peptide fragments; "spin" brands, such as amino acids that they contain nitroxide marks, and fatty acid derivatives; biotin; cross-linking agents containing carboxyl groups, such as diazoacetate, diazopyruvate, p-nitrophenyl-3-diazopyruvate, 2- (1,2-dithiolan-3-yl) acetate; N, N'-1,2-phenylenedimaleimide; N, N'-1,4-phenylene-dimaleimide. All the aforementioned derivatives have a carboxyl group which before of the enzymatic reaction is provided with one of the groups labile previously defined. So far it is in the case of the said fluorescent markers, not of the components complete carboxylics, but only one part, which is transferred on the amino component.

By joining the amino component and the carboxylic component a polypeptide or a polypeptide is formed selectively modified or analogues thereof, in which the C-terminal end of the amino component corresponds to the C-terminal end of the polypeptide, and the amino terminal end of the carboxylic component corresponds to amino end of the bound polypeptide under the influence of the enzyme or respectively of the marker or informative group introduced. The length of the synthesized or respectively modified polypeptide is At least two amino acids. Typically, the length of the polypeptide or protein obtained according to the invention has a size 1 to 1000 amino acids, preferably 30 to 1000, with more 50 to 600 preference and most preferably 100 to 300 amino acids.

The size of the amino component can be so Small as a single amino acid. An upper limit of the length of the amino component does not necessarily occur although the extreme final is determined, if it is determined, by specificity of the enzyme used as well as by kinetic considerations of the reaction, such as the diffusion rate of the amino component. Typical sizes of amine component are in this respect, from 1 to 1000 amino acids, of preferably 30 to 500 amino acids and more preferably, 30 to 250 amino acids It is however, within the framework of the present invention, that the length of the amino component is clearly greater, particularly in those versions of the procedure according to the invention in which a ligation of fragments of sequential peptides catalyzed by an enzyme or respectively a Protease, or a protein serves as an educt. The length is, of preference, a multiple of the length margins before cited. In this regard, it is within the framework of the present invention that the amino component is greater, equal or less than the component carboxylic, where as a criterion for this, it is used as a rule general the number of amino acids that make up the component amino or respectively, the carboxylic component.

It is preferred that the carboxylic component has a size of 1 to 1000 amino acids, preferably 30 to 500, still more preferably from 30 to 250 amino acids.

In the processes according to the invention, provided preferentially, which as an amino component, is employ peptides, mimetic substances of peptides and proteins with the unprotected N-terminal.

The reaction takes place preferably in pure ionic liquids such as p. eg, 4-methyl-N-butyl-pyridinium-tetrafluorborate , with none or only very little water content (typically less than 5%).

In another version of the invention, the proportion of ionic liquids in the reaction medium is 50-100% by volume, preferably 70-100 or 80-100% by volume, of 90-100% preference, equally preferred from 95 to 100% by volume or 97-99% by volume.

Mixtures of ionic liquids and solvents organic with and without proportion of water and other substances, such as p. eg, inorganic salts, also constitute reaction means in the meaning of the invention, where it does not matter if It is a solution or a suspension. As additives can in particular: inorganic salts, buffer components, reducing and oxidation agents, activators, modulators and enzyme inhibitors, surfactants, lipids, polymers for covalent or respectively adhesive protein immobilization (e.g. eg, polyethylene glycol, methoxypolyethylene glycol or carboxymethyl cellulose) and protein denaturing agents such as SDS (sodium dodecyl sulfate), urea or hydrochloride guanidino.

A decisive advantage of the use of mixtures of Solvents may consist of increasing or decreasing the solubility of the educts or enzymes or respectively in the influence of activity and enzyme specificity by the quantity, class and proportion of the solvent used additionally as ionic liquid.

According to the invention, they are preferably used with water, miscible organic solvents, when these are also mix with ionic liquids. On the other hand, they are those solvents also included according to the invention hydrophobic organics (e.g., hexane or octane), which are not miscible with water, in which the solvent mixture is as biphasic system According to the invention, the use of ionic liquids modified with alkyl groups hydrophobic, which either partially or totally, is mixed with hydrophobic organic solvents.

It is also preferred according to the invention to employ ionic liquids, in which the cations are, ions alkyl imidazolium, ions alkyl ammonium ions alkyl pyridinium and / or ions alkyl phosphonium, where the alkylation is complete in each case, that is to say none of the so-called heteroatoms is bound to a hydrogen, which are quaternized.

It is also preferred that the radicals alkyl of ionic liquids, branched or unbranched, and have 1-20 carbon atoms, preferably 1-20 carbon atoms, even more preferably, 4-6 carbon atoms. It is particularly preferred that at least one alkyl radical is methyl, ethylpropyl or butyl, in particular, butyl.

As anions of ionic liquids are used preferably according to the invention, chloride, bromide, chloro aluminate, nitrate, benzosulfonate, triflate (trifluorometansulfonate), tosylate and / or tetrafluorborate.

It is particularly preferred according to the invention, that as ionic liquids the salts of 1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, I 4-methyl-N-butyl-pyridinium, of which the corresponding one is particularly preferred tetrafluorborate

The synthesis of the carboxylic components with a labile group, preferably specific to an enzyme, can be carried out chemically by condensing the acyl radical of the carboxylic component with the corresponding labile group (or appropriate elements), or in polymeric supports, e.g. for example, by using sulfamyl butyrylamino-methyl- "Safety-Catch" resins (see R. Ingenito et al ., J. Am. Chem. Soc . 1999 , 121, 11369), or oxime resins (see V. Cerovsky, F. Bordusa, J. Peptide Res . 2000 , 55, 325, V. Cerovsky et al ., ChemBioChem 2000 , 2, 126) with simultaneous generation of ester or respectively amide, and cleavage of the peptide. As a separable nucleophile, the labile group itself containing the corresponding alcoholic, phenolic, mercapto or amino groups is used, as well as the esters or respectively amides of unprotected amino acids in Nα, or suitable precursors prepared previously. Alternatively, the synthesis of the carboxylic component can be carried out by genetic technical routes such as e.g. eg, by the use of synthesis of the intein-mediated polypeptide ester (MW Southworth et al ., Biotechniques (" Biotechnics ") 1999 , 27, 110). The adaptation of the labile group can be achieved by selecting the nucleophile in the cleavage of the intein or also after generating the ester by transesterification in solution.

The synthesis of peptide fragments used as amino components is carried out in solution or through the use of conventional Fmoc- synthesis protocols or Boc, in polymeric supports, routinely possible. Usually, synthesis in polymeric supports is preferred because to the advantages in the purification of intermediate products individual, compared to expensive synthesis in solution. Alternatively, the amino components can be obtained at from biological material or can be expressed by procedures of genetic techniques with exclusive isolation.

Products obtained synthetically or marked, can be separated and purified with the usual methods of the chemistry of peptides and proteins. Eventually, the groups Existing protection can be removed by procedures already known in the current state of the art.

According to a preferred version of the invention, a compound obtained according to steps a) to c) and possibly d), as an amino component, and another compound obtained according to steps a) to c) and possibly d), as a component carboxylic, to form a large polypeptide or protein of defined components, which may also present, according to the way of conducting the procedure, marker groups or informative

Unlike other procedures potentially employable biocatalysts, can be achieved by the use according to the invention of ionic liquids or mixtures of the themselves in combination with the use of enzymes and in particular proteases and peptidases, a high rate of synthesis, flexibility, synthesis efficiency and ease in handling.

The present invention is illustrated in view of the drawings and examples, from which others appear characteristics, versions and advantages of the invention.

In this regard, Figure 1 shows a spectrum of MALDI-ToF mass selected by use of the process according to the invention, and the biotinylated peptide obtained as described in example 4.

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Examples Example 1 Influence of the ionic liquid component 4-methyl-N-butyl-pyridinium tetrafluorborate on trypsin-catalyzed synthesis of di and tripeptides (Bz = benzoyl; Ogp = 4-guanidinophenyl ester)

1 ml of the reaction solution containing the mixtures indicated in table 1, of 4-methyl-N-butyl-pyridinium tetrafluoroborate, as well as 0.1 M of Hepes buffer pH 8.0, which contains 0.1 M of NaCl, and 0.01 M of CaCl2, 1.5% (v / v of 4-methylmorpholine, 2 mM of Bz-Phe-Ogp, 20 mM component Aminic, and 10 µM trypsin, is stirred at 25 ° C. After 30-120 minutes, the reaction solution is brought to pH 2 using 1 percent trifluoroacetic acid in methanol / water (1: 1, v / v). The di and tripeptide yields were determined analytically by HPLC, and are listed in the following table 1.

The synthesis of Bz-Phe-Ogp is achieved analogously to the synthesis protocol of M. Thormann et al ., Biochemistry 1999 , 38, 6056. The amine components used are commercially available products and are indicated in Table 1. Trypsin was purchased in Fluka (Switzerland).

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TABLE 1

one

Example 2 Influence of class and proportion of organic solvent added, on trypsin catalyzed synthesis of Bz-Phe-Leu-NH2 from Bz-Phe-Ogp and the H-Leu-NH2 in the ionic liquid 4-methyl-N-butyl-pyridinium tetra-fluoborate (Bz = benzoyl; OGp = 4-guanidinephenyl ester; MeOH = methanol; DMSO = dimethyl sulfoxide; DMF = dimethylformamide)

1 ml of reaction solution of 4-methyl-N-butyl-pyridinium tetrafluorborate, which contains 5% water and the proportion indicated organic solvent added, 1.5% (v / v) of 4-methylmorpholine, 2 mM of Bz-Phe-OGp, 20 mM component Aminic and 20, 40, 80, 200 µM trypsin (with a proportion increasing organic solvent added), stir at 25 ° C. After 30-120 minutes, the reaction solution is carried at pH 2 by 1 percent trifluoroacetic acid in methanol / water (1: 1, v / v). The yields were determined analytically by HPLC and are indicated in the following table 2.

TABLE 2

2

Example 3 Synthesis of polypeptides, catalyzed by trypsin, with sites specific cleaved enzymes, from Bz-Phe-OGp and polypeptides of different lengths and sequences, in the ionic liquid 4-methyl-N-butyl-pyridinium tetrafluorborate

The specific individual amino acid radicals of enzymes are enhanced in each case by bold
(Bz = benzoyl; OGp = 4-guanidinophenyl ester)

1 ml of reaction solution, which contains 4-methyl-N-butyl-pyridinium tetrafluoroborate, 5% water, 1.5% (v / v) of 4-methylmorpholine, 2 mM of Bz-Phe-OGp, 5 mM of components Amines and 10 µM trypsin are stirred at 25 ° C. For the purpose of solubility reactions were carried out with methanol as solvent additional organic, in which a proportion of the 20% as well as 50% (v / v) methanol. After 30-120 minutes the reaction solution is brought to pH 2 with 1 percent trifluoroacetic acid in methanol / water (1: 1, v / v). Polypeptide products identified by MALDI-ToF after its solution isolation of the reaction are listed in table 3 with the molecular mass calculated or respectively found in each case.

TABLE 3

3

Example 4 Introduction in the N-terminal of the group marker, biotin, trypsin catalyzed, in polypeptides with enzyme specific cleavage sites, from biotinyl-OGp and polypeptides of different lengths and sequences in the ionic liquid 4-methyl-N-butyl-pyridinium tetrafluorborate

The specific individual amino acid radicals of enzymes are enhanced in each case by bold
(Bz = benzoyl; OGp = 4-guanidinophenyl ester).

The reaction conditions correspond to those of example 3. Only the concentrations of the carboxylic component (biotinyl-OGp: 4 mM) and amino component (each peptide: 2 mM). The products of polypeptide identified from the reaction solution by MALDI-ToF, after their isolation they are indicated in table 4 with the corresponding molecular masses calculated or respectively found. The selectivity of the Biotinylation enzymatic reactions were determined by the use of acetylated peptide analogs in terminal N. The absence of product formation in these cases was assessed as a confirmation of biotinylation exclusively at the terminal N.

TABLE 4

4

The MALDI-ToF mass spectrum obtained from the biotinyl synthesis product - R IVDA R LEQV K AAGAY is represented as an example in Figure 1. The molar mass found from 1986.31 corresponds to the signal (M + H +) of the monoisotopic molar mass calculated 1985.05 of the peptide product.

Example 5 Selective introduction catalyzed with group chymotrypsin biotin marker in chicken albumin lysocin in the liquid ionic 4-methyl-N-butyl-pyridinium tetrafluorborate

Chymotrypsin has a specificity to relatively broad substrate, so the enzyme cleaves from preference aromatic esters of amino acids. The radicals of individual aromatic amino acids existing in lysozyme are enhanced in each case by bold (Hepes = acid N- [2-hydroxyethyl] piperazin-N '- [2-ethanesulfonic]; OGp = 4-guanidinophenyl ester).

Primary sequence of albumin lysozyme of chicken:

5

1 ml of reaction solution, which contains 4-methyl-N-butyl-pyridinium tetrafluorborate, 20% Hepes buffer (0.05 M, pH 8.0), 2 mM of biotinyl-OGp, 0.5 mM lysozyme and 10 µM of Chymotrypsin, stirred at 25 ° C. After 120 minutes he takes the reaction solution at pH 2 by the addition of acid 1 percent trifluoroacetic in methanol / water (1: 1, v / v). Mass molar found by MALDI-ToF after product insulation, 14589.06, of the synthesis product corresponds to the signal (M + H +) of the monoisotopic molar mass theoretically calculated from biotinylated lysozyme once.

The characteristics of the invention given to know in the description above, the claims as well as the drawings can be used both individually and also in any combination, for the achievement of the invention, in Different versions

Claims (26)

1. Procedure for peptide synthesis, mimetic substances of the peptides and / or proteins and / or for the selective modification in the N-terminal of peptides, substances mimetics of peptides and / or proteins, with the following Steps: a) preparation of an amino component, in where the amino component has at least one amino acid, b) preparation of a carboxylic component, in where the carboxylic component has a specific labile group of an enzyme in the carboxylic group, and the carboxylic component is a compound with at least one amino acid or a compound with, at least one marker or informational group, c) reaction of the amino component and the carboxylic component in a reaction medium which is composed of one or more ionic liquids, in the presence of a protease, peptidase and / or hydrolase, where between the component amine and the carboxylic component forms a peptide bond by cleavage of the labile group. 2. Method according to claim 1, which It also includes as a step: d) enrichment and / or isolation of the peptide, of the mimetic substance of peptides and / or of the protein obtained by procedures already known. 3. Method according to claim 1 or 2, characterized in that the amino component is a polypeptide or a protein. 4. Method according to one or more of the preceding claims, characterized in that the amino component has a size of 1 to 1000 amino acids, preferably 30 to 500, more preferably 30 to 250 amino acids.
two. 5. Method according to one or more of the preceding claims, characterized in that, the carboxyl end of the amino component is in an activated, protected or unprotected way. Method according to one or more of the preceding claims, characterized in that, the amino function Nα of the component amino is unprotected, where this amino function N? reacts with the carboxyl function of the carboxylic component. 7. Method according to one or more of the preceding claims, characterized in that, the carboxylic component is a polypeptide or a protein Method according to one or more of the preceding claims, characterized in that, the carboxylic component has a size of 1 to 1000 amino acids, preferably 30 to 500, more preferably 30 to 250 amino acids 9. Method according to one or more of the preceding claims, characterized in that, the carboxyl group of the carboxylic component form a carboxylic acid ester or an amide with the labile group of carboxylic acid. 10. Method according to one or more of the preceding claims, characterized in that, the labile group of the carboxylic component is choose from the group that includes the unsubstituted radicals or substituted: -O-alkyl, -O-aryl, -S-alkyl, radicals -S-aryl, -NH-alkyl, -NH-aryl, -N, N-dialkyl, -N, N-diaryl, and -N-aryl-N-alkyl. 11. Method according to claim 10, characterized in that, the labile group of the carboxylic component is substituted with one or more carboxylic acid radicals, sulfonic acid radicals, or sulfonates. 12. Method according to one or more of the preceding claims, characterized in that, The labile group is a radical 4-guanidinophenyl, 4-amidinophenyl, 4-guanidinophenylthio or 4-amidinophenylthio, or a compound of structure homologous to them. 13. Method according to one or more of the preceding claims, characterized in that, the labile group adapts to the specificity of the protease, peptidase and / or hydrolase used. 14. Method according to one or more of the preceding claims, characterized in that, the carboxylic component that contains the group Labil has the following structure: Y- (Xaa) n -R in where And it is a protection group of terminal N, or is H Xaa is an α-amino acid, any β-amino acid, or a derivative of the themselves, or a marker or informational group, R is a labile group, in particular, a group labile selected from the group comprising radicals without substitute or substituted, -O-alkyl, -O-aryl, -S-alkyl, radicals -S-aryl, 4-guanidinophenyl, 4-amidinophenyl, 4-guanidinophenylthio, 4-amidinophenylthio, which may be substituted with groups of sulfonic acids or sulphonates, as well as structural homologs thereof, n is an integer from 1 to 1000, preferably from 30 to 500, more preferably, from 30 to 250 15. Method according to one or more of the preceding claims, characterized in that, the carboxylic component is a marker group or informative, chosen from the fluorescent markers containing carboxyl groups, such as fluorescein, rhodamine, tetramethylrodamine, 2-aminobenzoic acid; marks of Isotopes containing carboxyl groups as amino acids or peptide fragments containing C 13 -, N 15 -, and O 17 -; "spin" brands containing carboxyl groups, such as amino acids containing the brand nitroxide and acid derivatives fatty cross-linking agents containing biotin-carboxyl groups such as diazoacetate, diazopyruvate, p-nitrophenyl-3-diazopyruvate; 2- (1,2-dithiolan-3-yl) acetate;  N, N'-1,4-phenylenedimaleimide. 16. Method according to one or more of the preceding claims, characterized in that, Steps a) to c) and eventually d), are performed two or more times to sequentially obtain a polypeptide or a protein, which has a marker or informational group. 17. Method according to one or more of the preceding claims, characterized in that, a compound is used as an amino component obtained according to steps a) to c) and eventually d), and as carboxylic component another compound obtained according to steps a) to c) and eventually d). 18. Method according to one or more of the preceding claims, characterized in that, the reaction medium has exclusively one or various ionic liquids. 19. Method according to one or more of claims 1 to 17, characterized in that, the reaction medium comprises one or more ionic liquids, and also water and / or an organic solvent and possibly usual additives. 20. Method according to one or more of the preceding claims, characterized in that, the proportion of ionic liquids in the middle of reaction is 50-100% by volume, preferably 70-100 or 80-100% by volume, with more preference 90-100% by volume, with equal preference of 95 to 100% by volume or 95-99% in volume. 21. Method according to one or more of the preceding claims, characterized in that, as cations of ionic liquids are used quaternized alkyl imidazolium ions, ions quaternized alkyl ammonium ions quaternized alkyl pyridinium and / or ions quaternized alkyl phosphonium. 22. Method according to claim 21, characterized in that, alkyl radicals of ionic liquids they are branched or unbranched and have 1-20 carbon atoms, preferably 2-10 atoms of carbon, and more preferably, 4-6 atoms of carbon. 23. Method according to one or more of the preceding claims, characterized in that, as ionic liquids, salts of 1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium I 4-methyl-N-butyl-pyridinium. 24. Method according to one or more of the preceding claims, characterized in that, as anions of ionic liquids are used chloride, bromide, chloroaluminate, nitrate, benzenesulfonate, triflate (trifluoromethanesulfonate), tosylate and / or tetrafluorborate 25. Method according to one or more of the preceding claims, characterized in that, as a protease a cysteine protease is used or serine protease 26. Use of a protease, peptidase and / or hydrolase for the synthesis and / or modification of N terminals, of peptides, mimetic substances of peptides and proteins, in where the peptide, the mimetic substance of the peptide and the protein or its species marked in terminal N, are obtained by ligation of an amino component and a carboxylic component, and the carboxylic component has a labile group.